Human Granulosa Cell Proliferation Research Articles

P-651 The role of FMR1-associated miR-323a and its impact on SMAD-signaling pathway in relation to the regulation of ovarian function

Abstract Study question Is FMR1 (Fragile-X-Mental Retardation 1 Gene)-targeting miR-323a related to poor ovarian response and what impact does it have on the folliculogenesis-regulating SMAD-signaling pathway? Summary answer In poor ovarian responders, miR-323a is significantly upregulated, FMRP – downregulated. Human granulosa cells, transfected with a miR-323a mimic, show an upregulation of SMAD proteins. What is known already Folliculogenesis is a complex process, regulated, among others, by FMR1 and its protein FMRP. MicroRNAs are involved in translational repression, controlling ovarian function and follicular development. miR-323a suppresses FMR1 in tumor cells, but their relationship has not been evaluated in the human ovary. miR-323a also interacts with components of SMAD pathways, which regulate cell proliferation, differentiation and apoptosis. SMADs belong to the downstream signaling of bone morphogenetic protein-receptor-2, and our previous data shows that its expression is affected by FMRP. Thus, researching miR-323a together with its target molecules may provide more insight into the pathogenesis of poor ovarian response. Study design, size, duration For ex vivo experiments, we investigated human granulosa cell (HGC) samples of poor (POR, N = 22) and normal (NOR, N = 32) ovarian responders, with an ongoing sample collection which began in 2013. Our in vitro experiments were performed on COV434 cell line, transfected with a microRNA-323a-3p mimic. RNA extraction and RT-qPCR were performed to determine the relative expression levels of the targets, with NOR serving as the control group ex vivo and non-transfected COV434 cells in vitro. Participants/materials, setting, methods 54 patients who underwent IVF/ICSI treatments were recruited and divided into two groups based on their ovarian response according to the Bologna criteria. HGCs were collected during oocyte retrieval, and COV434 cell line was used for miRNA mimic transfection experiments. Briefly, we performed RNA extraction, cDNA synthesis and TaqMan RT-PCR, as well as protein extraction and ELISA. The collected data was analysed using the 2–ΔΔCtmethod and Student’s t-test. Main results and the role of chance We found that miR-323a was significantly upregulated in HGCs of POR (p = 0.0324). FMR1 expression in this patient group showed a slight downregulation that did not reach statistical significance (p = 0.1054). Protein expression level analysis of 22 patients from this collective (NNOR=11, NPOR=10) demonstrated a significant downregulation of FMRP in POR (p = 0.0373), suggesting its participation in the pathogenesis of poor ovarian response. Additionally, in vitro upregulation of miR-323a resulted in altered expressions of SMAD-signaling pathway components. Namely, SMAD3 expression increased by 5.7 fold, SMAD4 – 1.6 fold, SMAD5 – 2.5 fold, and SMAD8 – 4.1 fold, compared to non-transfected COV434 cells, with SMAD3 results requiring further investigation due to an unexpected 2 fold elevation of its expression in the negative control (scramble) sample. FMR1 and SMAD1 expression levels remained stable between transfected cells and control samples. These findings show that FMR1-targeting miR-323a may play a critical role in regulating the SMAD pathways, thus affecting folliculogenesis, HGC proliferation and, ultimately, ovarian reserve, which is reduced in case of poor ovarian response. Limitations, reasons for caution Larger patient sample size is needed to confirm the current results. An age-related influence cannot be ruled out because of the existing difference between the groups. More repeats of miR-323a-3p mimic transfection and miR-323a-3p inhibitor transfection should be carried out to support the in vitro findings. Wider implications of the findings For the first time, we researched the expression levels of miR-323a in HGCs. Our results suggest that miR-323a, which, like FMRP, is downregulated in POR, is involved in the FMR1/FMRP regulatory loop, and its upregulation affects the SMAD-signaling pathway, deepening our understanding of the pathogenesis of poor ovarian response. Trial registration number not applicable

Human BM-MSC secretome enhances human granulosa cell proliferation and steroidogenesis and restores ovarian function in primary ovarian insufficiency mouse model

Primary ovarian insufficiency (POI) is defined as the loss of ovarian function before 40 years of age. It clinically manifests as amenorrhea, infertility, and signs of estrogen insufficiency. POI is frequently induced by chemotherapy. Gonadotoxic chemotherapy reagents damage granulosa cells, which are essential for follicular function and development. Our recently published studies demonstrated that intraovarian transplantation of human mesenchymal stem cells (hMSCs) can restore fertility in a chemotherapy-induced POI mouse model. However, the regenerative mechanism underlying the hMSC effect in POI mice is not fully understood. Here, we report that the hMSC secretome increased the proliferation of human granulosa cells (HGrC1). We showed by FACS analysis that treatment of HGrC1 cells with hMSC-conditioned media (hMSC CM) stimulates cellular proliferation. We also demonstrated that the expression of steroidogenic enzymes involved in the production of estrogen, CYP19A1 and StAR, are significantly elevated in hMSC CM-treated HGrC1 cells. Our data suggest that hMSC CM stimulates granulosa cell proliferation and function, which may explain the therapeutic effect of hMSCs in our chemotherapy-induced POI animal model. Our findings indicate that the hMSC secretome may be a novel treatment approach for restoring granulosa cell and ovarian function in patients with POI.

LncRNA LNC-GULP1-2:1 is involved in human granulosa cell proliferation by regulating COL3A1 gene

Long non-coding RNAs (lncRNAs) are defined as transcripts longer than 200 nucleotides that are not translated into protein. Many studies have identified the association between abnormal expression of lncRNAs and specific disease states, especially in tumor-related studies. However, the precise functions of these lncRNAs are not clear. In our previous study, both lnc-GULP1-2:1 and its potential target COL3A1 were significantly downregulated in ovarian cortical tissues of POI patients compared with normal control patients. The results of GO and KEGG analysis also showed that changes in extracellular matrix-related genes play an important role in the development of POI. Therefore, we speculate that lnc-GULP1-2:1 may participate in the development of ovarian follicles by regulating the COL3A1 gene. Basic research study. The lentiviral vectors were used to construct a KGN cell line with stable high expression of lnc-GULP1-2:1. Real-time PCR and western blot were used to analyze the expression of COL3A1 and lnc-GULP1-2:1. Cell proliferation was analyzed by using CCK-8 kits. The stable over-expression of lnc-GULP1-2:1 in KGN cells could significantly inhibit granulosa cell proliferation. Treatment of KGN cells with chemotherapeutic agents (cisplatin and paclitaxel) dose-dependently inhibited granulosa cell proliferation and promoted the expression of lnc-GULP1-2:1, whereas the cytokine TGFα, which promoted granulosa cell proliferation, inhibited the expression of lnc-GULP1- 2:1. These results suggest that lnc-GULP1-2:1 may be involved in proliferation regulation of granulosa cells. Further studies found that the protein levels of COL3A1 were significantly increased in KGN cells with stable over-expression of lnc-GULP1-2:1, indicating that lnc-GULP1-2:1 regulates the expression of COL3A1. Levels of lnc-GULP1-2:1 can be down-regulated by siRNAs (5 nM, 10 nM , and 20 nM) in different concentrations of COL3A1 in KGN cells, and silencing COL3A1 could also significantly inhibit KGN cell proliferation. The results of this study suggest that the involvement of lnc-GULP1-2:1 in the regulation of KGN granulosa cells may partly be mediated through the regulation of its target gene COL3A1. The regulation effect of lnc-GULP1-2:1 on granulosa cell proliferation was consistent with the occurrence of POI in clinical chemotherapeutic agents, suggesting that lnc-GULP1-2:1 may be involved in the regulation of granulosa cell function in POI patients. This is the first study to discover the regulation of lnc-GULP1-2:1 in ovarian granulosa cells, which is of great significance for the further investigation of lncRNA regulation in ovarian follicle development.

MiR-106a Increases Granulosa Cell Viability and Is Downregulated in Women With Diminished Ovarian Reserve.

Women with diminished ovarian reserve (DOR) have reduced fertility, cardiovascular events, and osteoporosis. Although differential microRNA (miRNA) expression has been described in several ovarian disorders, little is known about the role of miRNAs in the pathogenesis of DOR. Identify differentially expressed miRNAs in DOR and explore the role of miR-106a in human granulosa cell proliferation. miRNA microarray (n = 3) and quantitative reverse transcription polymerase chain reaction (n = 30) were used to examine miRNA expression in serum and granulosa cells from normal-cycling and women with DOR. Primary human granulosa cells were treated alone or in combination with miR-106a mimic, miR-106a inhibitor, apoptosis signal-regulating kinase 1 (ASK1) small interfering RNA (siRNA), or p38 mitogen-activated protein kinase (MAPK) inhibitor (SB203580) before assessment of cell viability and apoptosis. Western blot was used to measure ASK1 protein and phosphorylation/activation of p38 MAPK. Binding of miR-106a to ASK1 mRNA was examined by 3' untranslated region (3'UTR) luciferase analysis. Fifteen miRNAs were differentially expressed (n = 30), and miR-106a was downregulated in serum and granulosa cells of women with DOR. miR-106a mimic increased cell viability and attenuated apoptosis, whereas the converse occurred following treatment with miR-106a inhibitor. miR-106a suppressed ASK1 expression by directly targeting its 3'UTR. miR-106a inhibitor increased p38 MAPK phosphorylation/activation, and this effect was abolished by treatment with ASK1 siRNA. Whereas knockdown of ASK1 abolished the effects of miR-106a inhibitor on cell viability/apoptosis, pretreatment with SB203580 did not significantly alter the effects of miR-106a inhibitor. Downregulation of miR-106a may contribute to the pathogenesis of DOR by reducing granulosa cell viability and promoting apoptosis via enhanced ASK1 signaling.

FSH Regulates IGF-2 Expression in Human Granulosa Cells in an AKT-Dependent Manner

IGF-2 is highly expressed in the granulosa cells of human dominant ovarian follicles; however, little is known about the regulation of the IGF-2 gene or the interaction of IGF-2 and FSH during follicle development. To examine the mechanisms involved in the regulation of the IGF-2 gene by FSH and the interplay between FSH and IGF-2 during granulosa cell differentiation. Design, Setting, Patients, and Interventions: Cumulus granulosa cells were separated from cumulus-oocyte complexes isolated from the follicular aspirates of in vitro fertilization patients and cultured for in vitro studies. Protein and mRNA levels of IGF-2 and CYP19A1 (aromatase) were quantified using Western blot and quantitative real-time PCR. IGF-2 promoter-specific activation was determined by the amplification of alternative exons by PCR. Cell proliferation was assessed after treatment with FSH and/or IGF-2. FSH significantly enhanced IGF-2 expression after 8 hours of treatment and at low doses (1 ng/mL). Reciprocally, IGF-2 synergized with FSH to increase cell proliferation and the expression of CYP19A1. When IGF-2 activity was blocked, FSH was no longer able to stimulate CYP19A1 expression. Determination of IGF-2 promoter usage in human cumulus cells showed that the IGF-2 gene is driven by promoters P3 and P4. However, FSH exclusively increased P3 promoter-derived transcripts. Moreover, the FSH-induced stimulation of P3-driven IGF-2 transcripts was blocked by cotreatment with inhibitors of AKT or IGF-1 receptor (IGF-1R). The inhibitory effect of the IGF-1R inhibitor on FSH-induced IGF-2 mRNA accumulation was reversed by overexpression of a constitutively active AKT construct. FSH is a potent enhancer of IGF-2 expression in human granulosa cells. In return, IGF-2 activation of the IGF-1R and AKT is required for FSH to stimulate CYP19A1 expression and proliferation of granulosa cells. These findings suggest a positive loop interaction between FSH and IGF-2 that is critical for human granulosa cell proliferation and differentiation.

Negative Regulation of Human Granulosa Tumor Cell Survival by TGF-Beta.

Disruption to the regulatory pathways that govern granulosa cell proliferation, differentiation, and death during ovarian development and folliculogenesis can result in a variety of disorders, including granulosa cell tumor (GCT) formation. Candidate growth regulatory molecules produced within the ovary include several TGF-beta superfamily members, but the specific stimuli and intracellular signalling pathways which determine human granulosa cell proliferation and survival are still poorly understood. We have used a human ovarian cancer cell line of GCT origin, the KGN cells, to study TGF-beta superfamily actions. We have recently reported that this GCT line exhibited very little expression of the TGF-beta accessory receptor betaglycan, a TGF-beta and inhibin co-receptor which determines cellular sensitivity to these ligands. We hypothesized that GCTs would be refractory to the growth-inhibitory effects of TGF-beta and/or inhibin and that overexpression of betaglycan would restore responsiveness to TGF-beta mediated growth inhibition. To test this hypothesis, KGN cells were stably transfected with a wildtype betaglycan expression construct or the empty vector as a control. Overexpression of betaglycan in the KGN cells restored sensitivity to TGF-beta and inhibin in luciferase reporter assays but had little effect on cell proliferation, cell cycle progression, or death in either GCT cell line as determined by flow cytometry. Furthermore, treatment with either exogenous TGF-beta2 or inhibin A, INHA knockdown, or neutralisation of endogenous TGF-betas also did not significantly affect cell survival and proliferation. As the GCT cells are tumour cell lines which exhibit constitutively active NF-kappaB, which is a pro-survival factor, we pre-treated the cells with the chemical NF-kappaB inhibitor, BAY11-7082. Under these conditions, overexpression of betaglycan resulted in a decrease in proliferation (15%; p<0.05) and an increase in apoptosis (300% p<0.05). The addition of TGF-beta1 and TGF-beta2 dose-dependently decreased cell viability, and this effect was enhanced by the presence of betaglycan. However, knockdown of INHA expression did not affect cell viability even in the presence of BAY11-7082. Following treatment with exogenous TGF-beta2, INHA gene knockdown, or a combination of the two, total cell lysates were extracted from betaglycan- and vector-transfected GCT cells. The relative levels of activation of specific pathways were determined by western blot analyses using activation-specific antibodies against SMAD2, SMAD3, and SMAD1/5. This analysis revealed that overexpression of betaglycan in the GCT cells increased both the basal and TGF-beta2-induced levels of activated SMAD3, with a smaller effect observed on activated SMAD2. Collectively, these data establish the TGF-beta-SMAD2/3-betaglycan signalling pathway as an important regulator of granulosa cell viability and suggest that deficiency of this pathway and/or inhibition by NF-kappaB contributes to dysregulated granulosa cell growth and cancer. Supported by the NHMRC of Australia (RegKeys 494802; 441101; 388904) and Victorian Government Infrastructure funds. (platform)

Insulin attenuates IGF-1-induced luteinized granulosa cell proliferation with an increase in PTEN

OBJECTIVE: The influence of insulin on folliculogenesis in women with polycystic ovary syndrome (PCOS), which ofthen shows insulin resistance, has not been fully investigated. To assess the induction of PTEN expression with insulin treatment and effects of PTEN on IGF-1-induced granulosa cell proliferation as well as the correlation of PTEN levels with the concentration of insulin in follicular fluid in PCOS and non-PCOS patients. DESIGN: Assay and in-vitro experiments using human granulosa cells and follicular fluids, and analysis of the correlation with clinical data. MATERIALS AND METHODS: A cell proliferation assay, real-time RT-PCR and Western blotting for PTEN, Akt and ERK1/2 were conducted in primary cultured granulosa cells under IGF-1 stimulation with or without insulin pretreatment. Phosphorylation of Akt and ERK1/2 was also determined by Western blotting. We also measured the insulin concentration in follicular fluid and the levels of PTEN expression in granulosa cells collected at the time of oocyte retrieval of IVF in PCOS and non-PCOS patients. RESULTS: PTEN expression was induced by insulin. Pretreatment with insulin attenuated the IGF-1-induced Akt phosphorylation and cell proliferation, but not ERK1/2 phosphorylation. Suppression of insulin-induced PTEN expression using siRNA recovered IGF-1-induced Akt phosphorylation. PTEN levels in granulosa cells, which tended to be higher in PCOS patients, were correlated with the insulin concentration in follicular fluid. CONCLUSIONS: PTEN may influence the proliferation of human granulosa cells as well as disturbance of follicular growth in PCOS patients.

Effects of growth differentiation factor 9 on cell cycle regulators and ERK42/44 in human granulosa cell proliferation

GDF-9 stimulates granulosa cell proliferation and plays important roles during folliclogenesis. However, its molecular mechanisms are still far from clear, particularly its roles in human granulosa cells around the periovulatory stage. Therefore, we investigated the effects of GDF-9 on cell cycle distribution, regulatory molecules, and signaling pathways involved in human luteinized granulosa (hLG) cells in vitro. Primary cultures of hLG cells obtained from women undergoing IVF and treated with and without recombinant GDF-9 were evaluated with and without a specific inhibitor to activin receptor-like kinase 5 (ALK5; SB-431542), ERK42/44 (PD-098059), or Smad3 (SIS3). Cell proliferation, cell cycle distribution, mRNA expression, and protein expression of relevant cell cycle molecules were determined by [(3)H]thymidine incorporation, flow cytometry, quantitative PCR, and immunoblotting, respectively. GDF-9 stimulated [(3)H]thymidine incorporation, enhanced cell transition from G(0)/G(1) to S and G(2)/M phases (whereas both SB-431542 and PD-098059 attenuated these changes), increased mRNA and protein expression of cyclin D(1) and E, and decreased those of the cyclin-dependent kinase (CDK) inhibitors p15(INK4B) and p16(INK4A). GDF-9 also activated Rb protein (a critical G(1) to S-phase regulator), ERK42/44, and Smad3. PD-098059 blocked Rb protein phorsphorylation and the increase in cyclin D(1) and E but not the decrease in p15(INK4B) and p16(INK4A) induced by GDF-9. In contrast, SIS3 reversed the decrease in p15(INK4B) and p16(INK4A) but not the increase in cyclin D(1) and E induced by GDF-9. GDF-9 stimulates hLG cell proliferation by stimulating cyclin D(1) and E and suppressing p15(INK4B) and p16(INK4A) via both Smad-dependent and Smad-independent pathways.

Insulin Attenuates the Insulin-Like Growth Factor-I (IGF-I)-Akt Pathway, not IGF-I-Extracellularly Regulated Kinase Pathway, in Luteinized Granulosa Cells with an Increase in PTEN

Insulin resistance is considered as part of the pathogenesis of polycystic ovary syndrome (PCOS), and PCOS patients often show hyperinsulinemia. The influence of insulin on folliculogenesis in women with PCOS has not been fully investigated. Our objective was to assess the induction of phosphatase and tensin homolog deleted on chromosome 10 (PTEN) expression with insulin treatment and effects of PTEN on IGF-I-induced granulosa cell proliferation as well as the correlation of PTEN levels with the concentration of insulin in follicular fluid in PCOS and non-PCOS patients. DESIGN, SETTING, PATIENTS, AND MAIN OUTCOME MEASURES: A cell proliferation assay, real-time RT-PCR, and Western blotting for PTEN, Akt, and ERK1/2 were conducted in primary cultured granulosa cells under IGF-I stimulation with or without insulin pretreatment. Phosphorylation of Akt and ERK1/2 was also determined by Western blotting. We also measured the insulin concentration in follicular fluid and the levels of PTEN expression in granulosa cells collected at the time of oocyte retrieval of in vitro fertilization in PCOS (n = 13) and non-PCOS patients (n = 37). PTEN expression was induced by insulin. Pretreatment with insulin attenuated the IGF-I-induced Akt phosphorylation and cell proliferation but not ERK1/2 phosphorylation. A phosphatidylinositol 3-kinase inhibitor, LY294002, inhibited the IGF-I-induced cell proliferation. Suppression of insulin-induced PTEN expression using small interfering RNA recovered IGF-I-induced Akt phosphorylation. PTEN levels in granulosa cells, which tended to be higher in PCOS patients, were correlated with the insulin concentration in follicular fluid. PTEN may influence the proliferation of human granulosa cells as well as disturbance of follicular growth in PCOS patients.

Insulin Attenuates the Insulin-Like Growth Factor-I (IGF-I)-Akt Pathway, not IGF-I-Extracellularly Regulated Kinase Pathway, in Luteinized Granulosa Cells with an Increase in PTEN

Insulin resistance is considered as part of the pathogenesis of polycystic ovary syndrome (PCOS), and PCOS patients often show hyperinsulinemia. The influence of insulin on folliculogenesis in women with PCOS has not been fully investigated. Our objective was to assess the induction of phosphatase and tensin homolog deleted on chromosome 10 (PTEN) expression with insulin treatment and effects of PTEN on IGF-I-induced granulosa cell proliferation as well as the correlation of PTEN levels with the concentration of insulin in follicular fluid in PCOS and non-PCOS patients. A cell proliferation assay, real-time RT-PCR, and Western blotting for PTEN, Akt, and ERK1/2 were conducted in primary cultured granulosa cells under IGF-I stimulation with or without insulin pretreatment. Phosphorylation of Akt and ERK1/2 was also determined by Western blotting. We also measured the insulin concentration in follicular fluid and the levels of PTEN expression in granulosa cells collected at the time of oocyte retrieval of in vitro fertilization in PCOS (n = 13) and non-PCOS patients (n = 37). PTEN expression was induced by insulin. Pretreatment with insulin attenuated the IGF-I-induced Akt phosphorylation and cell proliferation but not ERK1/2 phosphorylation. A phosphatidylinositol 3-kinase inhibitor, LY294002, inhibited the IGF-I-induced cell proliferation. Suppression of insulin-induced PTEN expression using small interfering RNA recovered IGF-I-induced Akt phosphorylation. PTEN levels in granulosa cells, which tended to be higher in PCOS patients, were correlated with the insulin concentration in follicular fluid. PTEN may influence the proliferation of human granulosa cells as well as disturbance of follicular growth in PCOS patients.

Ovarian KCNQ channels and regulation by non-neuronal acetylcholine: Modulation of human granulosa cell proliferation and steroidogenesis

Ovarian KCNQ channels and regulation by non-neuronal acetylcholine: Modulation of human granulosa cell proliferation and steroidogenesis

P-781: Proliferation of human granulosa cells from stimulation cycles could be a positive indicator for proper ovarian hyperstimulation

Proper ovarian hyperstimulation for oocytes with good quality is one of the main contributors to the success of human in vitro fertilization-embryo transfer (IVF-ET) program. So, identification of markers, which imply proper stimulation protocol for patients, should be helpful on the improvement of human ART program. Several characteristics of human granulosa cells (GCs) obtained from IVF-ET program have been studied for estimating the outcome. The present study was performed to find the relationship between proliferating level of cultured GCs and the outcome of IVF-ET (embryo quality and pregnancy rate).

The role of endothelin-1 in regulating human granulosa cell proliferation and steroidogenesis in vitro

The role of endothelin-1 in regulating human granulosa cell proliferation and steroidogenesis in vitro

Insulin-like growth factor-II stimulates human granulosa-luteal cell proliferation in vitro

To determine the effect of insulin-like growth factor-II (IGF-II) on human granulosa cell proliferation in vitro. Thymidine incorporation and cell count were determined to assess human granulosa cell proliferation. The study was performed in an academic research environment. Granulosa cells were obtained from women undergoing IVF procedures. Granulosa cells were dispersed enzymatically and cultured in both serum-containing and serum-free medium. Granulosa cell proliferation in basal conditions and after stimulation with IGF-II given alone or in combination with basic fibroblast growth factor (basic FGF). Treatment of human granulosa cells with IGF-II for 24 hours significantly increased thymidine incorporation. Furthermore, this mitogenic effect was confirmed when granulosa cells were cultured in presence of IGF-II for 7 to 10 days and their proliferation was evaluated by cell counting. The association of IGF-II and basic FGF did not result in a synergistic or additive effect. As already demonstrated in other species, IGF-II also is able to stimulate human granulosa cell proliferation in vitro. Thus, it might be considered among the intraovarian modulators of granulosa cell physiology.

The role of progesterone in regulating human granulosa cell proliferation and differentiation in vitro

The role of progesterone in regulating human granulosa cell proliferation and differentiation in vitro

The role of progesterone in regulating human granulosa cell proliferation and differentiation in vitro.

To further elucidate the role of progesterone in regulating granulosa cell function, human granulosa and luteal cells were obtained from follicular aspirates of women undergoing in vitro fertilization and placed in culture. Cells plated at 5 x 10(3) cells/mL doubled after 3 days. In contrast, cells plated at 50 x 10(3) cells/mL did not proliferate, but differentiated, secreting high levels of progesterone. Cells plated at 5 x 10(3) cells/mL and cultured with spent medium from cells plated at 50 x 10(3) cells/mL did not increase in number over 3 days of culture. The growth-inhibiting action of the spent medium was removed by either RU 486 (a progesterone receptor antagonist) or charcoal extraction, but not by heat inactivation. The addition of progesterone to fresh medium also prevented cell proliferation. Progesterone's ability to inhibit cell division was attenuated by either RU 486 or aminoglutethamide, which blocked progesterone synthesis. Further, epidermal growth factor (EGF) stimulated cell proliferation, and continuous exposure to progesterone blocked EGF-induced mitosis. When progesterone was added 2 h after EGF, it did not block EGF-stimulated cell proliferation. Progesterone also increased the percentage of granulosa cells and decreased the percentage of large luteal cells present after 3 days of culture, indicating that progesterone inhibited differentiation. Progesterone's effect on differentiation was dose dependent, reversible, and could be overridden by hCG or 8-bromo-cAMP. These observations suggest that progesterone acts directly on granulosa cells through its receptor to inhibit mitosis and that progesterone mediates its antiproliferative effects within 2 h of mitotic stimulation. Progesterone also blocks differentiation, but this effect of can be overcome by hCG or cAMP analogs. These data indicate that progesterone plays a major role in controlling the number of luteal cells that ultimately develop within a corpus luteum by regulating both granulosa cell proliferation and differentiation.

Progesterone as an autocrine/paracrine regulator of human granulosa cell proliferation.

The present study examined the role of progesterone in regulating human granulosa cell proliferation. Human granulosa and luteal cells were obtained from follicular aspirates of women undergoing in vitro fertilization. Cells were plated at 5, 10, or 50 x 10(3) cells/mL and cultured for up to 6 days. At specific times, cells were harvested and assessed for cell number and morphology. The medium was assayed for progesterone. Cells plated at 50 x 10(3) cells/mL did not increase in number after 3 or 6 days of culture, but rapidly differentiated, secreting high amounts of progesterone (> or = 320 nmol/L). Conversely, cells plated at 5 x 10(3) or 10 x 10(3) cells/mL doubled in number over the first 3 days of culture and subsequently differentiated. The addition of 100 ng/mL progesterone or more to the medium inhibited proliferation. Aminoglutethamide blocked progesterone secretion and increased the number of cells present after 3 days of culture. The antiproliferative effects of progesterone were not mimicked by estradiol, testosterone, dihydrotestosterone, or dexamethasone and could not be overridden by epidermal growth factor, a potent mitogen. These data suggest that progesterone plays an autocrine/paracrine role in regulating granulosa cell proliferation.

Progesterone as an autocrine/paracrine regulator of human granulosa cell proliferation

Progesterone as an autocrine/paracrine regulator of human granulosa cell proliferation

Insulin-like growth factor binding protein-1 inhibits the DNA amplification induced by insulin-like growth factor I in human granulosa—luteal cells

In order to study the effects of insulin-like growth factor (IGF-I) and insulin-like growth factor binding protein (IGFBP-1) on human granulosa cell proliferation after in vitro fertilization, cells were obtained after oocyte retrieval and cultured in the presence or absence of graded amounts of recombinant IGF-I, purified IGFBP-1 and [3H]thymidine. Physiological concentrations of IGF-I (2-200 ng/ml) were found to stimulate [3H]thymidine incorporation into the cells in a concentration-dependent manner. Half-maximal stimulation of [3H]thymidine incorporation was obtained with 10 ng/ml exogenous IGF-I, which was chosen for suppression experiments with graded amounts of purified IGFBP-1. Suppression of IGF-stimulated thymidine incorporation was observed when 200 ng/ml or more of IGFBP-1 was added to the culture medium. The same concentration of IGFBP-1 also markedly inhibited binding of [125I]iodotyrosyl IGF-I to the cells. It is concluded that: (i) after a refractory period, granulosa cells from hyperstimulated follicles retained their mitogenic activity; (ii) IGF-I is capable of stimulating DNA amplification in granulosa cells; and (iii) IGFBP-1 inhibits the IGF-I stimulated proliferation in these cells. In view of our previous studies showing that IGFBP-1 is synthesized by the granulosa cells as they luteinize, the present results suggest that IGFBP-1 is one of the endogenous factors locally regulating the growth and differentiation of granulosa cells.